血管平滑肌钙激活钾通道及其调节机制

在所有离子通道中，钾通道种类最多。就血管平滑肌细胞（ｖａｓｃｕｌａｒｓｍｏｏｔｈｍｕｓｃｌｅｃｅｌ，ＶＳＭＣ）而言，目前正式命名的就有４大类即电压依赖性钾通道（Ｋｖ）、钙激活的钾通道（ＫＣａ）、内向整流钾通道（ＫＩＲ）和ＡＴＰ敏感性钾通道（ＫＡＴＰ）...     

ATP敏感性钾通道分子结构和功能的组织特异性研究进展

ＡＴＰ敏感性钾通道（ＫＡＴＰ）通过与细胞代谢和生物电活动相偶联,在许多组织发挥着重要的生理和病理生理学功能。ＫＡＴＰ由内向整流钾通道（Ｋｉｒ）和ＡＴＰ结合蛋白两部分组成,前者形成离子通道,后者决定着ＫＡＴＰ的功能。Ｋｉｒ和ＡＴＰ结合蛋白又分多种亚型,两者不同亚型间的相互偶联导致了不同组织ＫＡＴＰ分子结构的多样性,分子结构的不同又决定了不同组织ＫＡＴＰ特性和功能的复杂性。本文对不同组织ＫＡＴＰ的分子结构、生理学功能、病理生理学和药理学特性进行了综述。     

Characterization of subtype of α_1-adrenoceptor mediating vasoconstriction in perfused rat mesenteric vascular bed

目的：研究去甲肾上腺素（ＮＥ）介导大鼠肠系膜血管床（ＭＶＢ）收缩的α１肾上腺素受体（α１ＡＲ）亚型．方法：用灌流大鼠ＭＶＢ标本收缩功能实验和克隆细胞放射配体结合实验测定α１ＡＲ亚型选择性拮抗剂ｐＡ２和ｐＫｉ,并作相关分析．结果：α１ＡＡＲ选择性拮抗剂ＲＳ１７０５３、ＷＢ４１０１、５ＭＵ及α１ＤＡＲ选择性拮抗剂ＢＭＹ７３７８的ｐＡ２分别为８９８±０２８,９１６±０２０,８．６９±００２和６０３±０２６,Ｓｃｈｉｌｄ作图斜率值与１０差别无显著性．其ｐＡ２值与α１ＡＡＲ的ｐＫｉ相关系数为０９７,与α１Ｂ和α１ＤＡＲ的相关系数分别为０５２和００４．结论：介导外源性ＮＥ收缩大鼠ＭＶＢ的功能性受体为α１ＡＡＲ     

N-甲基小檗胺对豚鼠单一心室肌细胞ATP敏感钾电流的影响

目的　研究Ｎ 甲基小檗胺对豚鼠单一心室肌细胞ＡＴＰ敏感钾电流的作用。方法　膜片钳制技术全细胞记录模式。结果　Ｎ 甲基小檗胺抑制心室肌细胞ＡＴＰ敏感钾电流（ＩＫＡＴＰ）且具浓度依赖关系。指令电位为０ｍＶ时,３种浓度Ｎ 甲基小檗胺（０－１,１,１０μｍｏｌ·Ｌ－１）可使ＩＫＡＴＰ分别由给药前的（０－４６±０－０９）ｎＡ,（０－４３±０－１５）ｎＡ和（０－４７±０－１０）ｎＡ减少至给药后的（０－３７±０－０７）ｎＡ（ｎ＝４,Ｐ＜０－０５）,（０－２９±０－１８）ｎＡ（ｎ＝５,Ｐ＜０－０５）和（０－２１±０－０７）ｎＡ（ｎ＝４,Ｐ＜０－０５）。其抑制率分别为１８－８１％±６－１６％（Ｐ＜０－０５）,４１－４７％±２４－０５％（Ｐ＜０－０５）和５５－００％±１２－９４％（Ｐ＜０－０５）。其它指令电位下的ＩＫＡＴＰ的改变也符合此趋势。结论　Ｎ 甲基小檗胺阻断豚鼠心肌细胞ＡＴＰ敏感Ｋ＋钾通道。     

吡那地尔对豚鼠回肠壁副交感节后神经元和回肠平滑肌的影响

ＡＴＰ敏感性钾通道（ＡＴＰ－ｓｅｎｓｉｔｉｖｅｐｏｔａｓｓｉｕｍｃｈａｎｎｅｌ,ＫＡＴＰ）广泛分布于心血管系统,内分泌腺（主要指胰腺）,中枢神经系统等．吡那地尔（ｐｉｎａｃｉｄｉｌ）为高选择性ＫＡＴＰ激活剂［１］,于１９８７年在丹麦上市,用于治疗原发...     

氯化钾泡腾颗粒的药代动力学研究

２０名健康自愿受试者分别于规定时间口服氟化钾泡腾颗粒（ＴＰＥＧ）和１０％氯化钾（ＫＣｌ）液，用原子吸收分光光度法测定血清Ｋ＋浓度，以二室模型拟合，在ＴＢＭ机上计算药动学参数。结果表明：实验组与对照组氯化钾的Ｋａ、ｔ（１／２）Ｋａ、α、ｔ（１／２）α、ＡＵＣ（０－∞）很接近，两者的Ｋ（２１）／Ｋ（１２）之比值分别为４．８５９和１．２２２，ＡＵＣ（０－∞）分别为１９７．１９０ｍｇ／ｍｌ·ｈ和２００．５８５ｍｇ／ｍｌ·ｈ，经ＳｔｕｄｅｎｔＴ检验无显著差异（ｐ＞０．０５），Ｆ＝９８．３％，证明ＴＰＥＧ有较好的补血钾效果。     

Dual effects of tetrandrine on calcium-activated potassium channels in pulmonary artery smooth muscle cells

目的：研究粉防己碱（Ｔｅｔ）对大鼠肺动脉平滑肌细胞钙激活钾（ＫＣａ）通道的影响．方法：内面朝外膜片箝单通道记录法．结果：Ｔｅｔ７５和１５μｍｏｌ·Ｌ－１使ＫＣａ的开放概率由０２５１±００１２增加到０３４０±００１３和０４１５±００１１（Ｐ＜００１）．关闭时间由（６１±１５）ｍｓ缩短到（３３±１０）和（２８±１１）ｍｓ（Ｐ＜００１）．Ｔｅｔ３０μｍｏｌ·Ｌ－１使开放概率和开放时间分别降低到（０１１４±０００８）和（１４７±００９）ｍｓ（Ｐ＜０．０１）．结论：Ｔｅｔ对大鼠肺动脉平滑肌细胞ＫＣａ通道有双重作用．     

Antisense oligodeoxynucleotides downregulated c sis mRNA expression to inhibit proliferation of vascular smooth muscle cells 1

目的：研究ｃｓｉｓ反义寡脱氧核苷酸（ＯＤＮ）对ｃｓｉｓ表达及血管平滑肌细胞（ＶＳＭＣ）增殖抑制效应．方法：用合成ｃｓｉｓ正、反义ＯＤＮ培养ＶＳＭＣ;用液闪测定［３Ｈ］ＴｄＲ掺入并细胞计数,观察细胞增殖;用逆转录ＰＣＲ,评价ｃｓｉｓ表达．结果：ｃｓｉｓ反义ＯＤＮ２,４,６,８,１０μｍｏｌ·Ｌ－１抑制ＶＳＭＣ（１０３％±０７％,２２６％±０９％,３１０％±１１％,３５４％±０９％,４３３％±１２％）和降低［３Ｈ］ＴｄＲ掺入（６８％±０３％,９７％±０７％,２９０％±０６％,３２０％±０７％,５０６％±１３％）呈有剂量依赖性．反义ＯＤＮ１０μｍｏｌ·Ｌ－１培养细胞４ｄ,最大抑制率达６０３％±１０％,［３Ｈ］ＴｄＲ掺入降低５６３％±０９％,ｃｓｉｓｍＲＮＡ表达明显降低;而正义ｃｓｉｓＯＤＮ对ＶＳＭＣ无抑制,细胞数和［３Ｈ］ＴｄＲ掺入及ｃｓｉｓｍＲＮＡ水平与对照无差异．结论：ｃｓｉｓ反义ＯＤＮ明显下调ｃｓｉｓｍＲＮＡ表达,显著抑制ＶＳＭＣ增殖     

Stimulation of locus coeruleus increases arterial pressure in rabbits

目的：研究电刺激和化学刺激兔蓝斑（ＬＣ）对动脉血压（ＡＰ）和肾交感神经传出活动（ＲＳＡ）的影响．方法：电刺激ＬＣ，ＬＣ微量注射ＬＧｌｕ、盐酸吗啡、ＧＡＢＡ、电解毁损ＬＣ，记录ＡＰ和ＲＳＡ．结果：电刺激ＬＣ和ＬＣ注射ＬＧｌｕ均引起ＡＰ升高（分别为１３５±０３ｖｓ１９５±０８ｋＰａ和１３８±０４ｖｓ１７５±０８ｋＰａ）和ＲＳＡ增加．ＬＣ注射吗啡、ＧＡＢＡ对ＡＰ和ＲＳＡ无明显影响．电解毁损ＬＣ后电刺激ＬＣ区、ＬＣ区注射ＬＧｌｕ对ＡＰ和ＲＳＡ无明显影响．结论：兔ＬＣ兴奋引起ＡＰ升高和ＲＳＡ增加，但ＬＣ不是ＡＰ和ＲＳＡ的紧张性中枢．     

Differential effects of pinacidil,cromakalim,and NS 1619 on electrically evoked contractions in rat vas deferens

目的：本文研究新近研制的ＡＴＰ敏感性钾通道开放剂，吡那地尔（Ｐｉｎ）和ｃｒｏｍａｋａｌｉｍ（Ｃｒｏ），以及钙离子激活性钾通道开放剂ＮＳ１６１９对电场刺激所致大鼠输精管收缩的作用．方法：利用电场刺激（０．３Ｈｚ，１ｍｓ，６０Ｖ）反复性引致输精管单相性收缩．结果：Ｐｉｎ和Ｃｒｏ浓度依赖性减低电刺激收缩．格列本脲（Ｇｌｉ）而非ｃｈａｒｙｂｄｏｔｏｘｉｎ拮抗上述两药的舒张作用．Ｐｉｎ右移去甲肾上腺素的浓度－收缩曲线，同时降低最高收缩反应．Ｇｌｉ抵消Ｐｉｎ的作用．Ｃｈａｒｙｂｄｏｔｏｘｉｎ而非Ｇｌｉ减低ＮＳ１６１９的平滑肌舒张作用．结论：ＡＴＰ敏感性和钙离子激活性钾通道参与调节输精管平滑肌的收缩性．     

ATP敏感钾通道亚单位在大鼠组织中的表达

目的　研究ATP敏感钾通道 (KATP)亚单位在大鼠组织中的表达。方法　逆转录多聚酶链式反应 (RT PCR)检测通道mRNA的表达。结果　左心室有Kir 6 1,Kir 6 2和SUR 2A的表达 ,大脑皮层中Kir 6 1,Kir 6 2 ,SUR 1,SUR2B均有表达 ,主动脉平滑肌有Kir 6 1,Kir 6 2 ,SUR 2B的表达 ,膀胱平滑肌中有Kir 6 1,Kir 6 2 ,SUR 2B的表达。结论　各组织的KATP组成有所不同 ,Kir 6 1、Kir 6 2和SUR 2B亚单位在多组织中广泛表达。     

Mechanism of terfenadine block of ATP-sensitive K(+) channels

The ATP-sensitive K(+) (K(ATP)) channel is a complex of a pore-forming inwardly rectifying K(+) channel (Kir6.2) and a sulphonylurea receptor (SUR). The aim of the present study was to gain further insight into the mechanism of block of K(ATP) channels by terfenadine. Channel activity was recorded both from native K(ATP) channels from the clonal insulinoma cell line RINm5F and from a C-terminal truncated form of Kir6.2 (Kir6.2Delta26), which - in contrast to Kir6.2 - expresses independently of SUR. Kir6.2Delta26 channels were expressed in COS-7 cells, and enhanced green fluorescent protein (EGFP) cDNA was used as a reporter gene. EGFP fluorescence was visualized by a laser scanning confocal microscope. Terfenadine applied to the cytoplasmic side of inside-out membrane patches concentration-dependently blocked both native K(ATP) channel and Kir6.2Delta26 channel activity, and the following values were calculated for IC(50) (the terfenadine concentration causing half-maximal inhibition) and n (the Hill coefficient): 1.2 microM and 0.7 for native K(ATP) channels, 3.0 microM and 1.0 for Kir6. 2Delta26 channels. Terfenadine had no effect on slope conductance of either native K(ATP) channels or Kir6.2Delta26 channels. Intraburst kinetics of Kir6.2Delta26 channels were not markedly affected by terfenadine and, therefore, terfenadine acts as a slow channel blocker on Kir6.2Delta26 channels. Terfenadine-induced block of Kir6. 2Delta26 channels demonstrated no marked voltage dependence, and lowering the intracellular pH to 6.5 potentiated the inhibition of Kir6.2Delta26 channels by terfenadine. These observations indicate that terfenadine blocks pancreatic B-cell K(ATP) channels via binding to the cytoplasmic side of the pore-forming subunit. The presence of the pancreatic SUR1 has a small, but significant enhancing effect on the potency of terfenadine.     

Inhibition of recombinant K(ATP) channels by the antidiabetic agents midaglizole,LY397364 and LY389382

Most imidazolines inhibit ATP-sensitive K(+) (K(ATP)) channels. Since these drugs are potentially clinically relevant insulin secretagogues, it is important to know whether extrapancreatic K(ATP) channels are targeted. We examined the effects of three imidazoline-derived antidiabetic drugs on the cloned K(ATP) channel, expressed in Xenopus laevis oocytes, and their specificity for interaction with the pore-forming Kir6.2 or the sulphonylurea receptor (SUR) 1 subunit. Midaglizole, LY397364 and LY389382 blocked Kir6.2deltaC currents with IC(50) of 3.8, 6.1 and 0.7 microM, respectively. The block of Kir6.2/SUR1 currents by LY397364 and LY389382 was best fit by a two-site model, suggesting that these drugs also interact with SUR1. However, since all three drugs interact with the Kir6.2 subunit, and Kir6.2 forms the pore of extrapancreatic K(ATP) channels, these drugs are unlikely to be specific for the beta-cell.     

A transmembrane domain of the sulfonylurea receptor mediates activation of ATP-sensitive K(+) channels by K(+) channel openers.

ATP-sensitive K(+) (K(ATP)) channels are a complex of an ATP-binding cassette transporter, the sulfonylurea receptor (SUR), and an inward rectifier K(+) channel subunit, Kir6.2. The diverse pharmacological responsiveness of K(ATP) channels from various tissues are thought to arise from distinct SUR isoforms. Thus, when assembled with Kir6. 2, the pancreatic beta cell isoform SUR1 is activated by the hyperglycemic drug diazoxide but not by hypotensive drugs like cromakalim, whereas the cardiac muscle isoform SUR2A is activated by cromakalim and not by diazoxide. We exploited these differences between SUR1 and SUR2A to pursue a chimeric approach designed to identify the structural determinants of SUR involved in the pharmacological activation of K(ATP) channels. Wild-type and chimeric SUR were coexpressed with Kir6.2 in Xenopus oocytes, and we studied the resulting channels with the patch-clamp technique in the excised inside-out configuration. The third transmembrane domain of SUR is found to be an important determinant of the response to cromakalim, which possibly harbors at least part of its binding site. Contrary to expectations, diazoxide sensitivity could not be linked specifically to the carboxyl-terminal end (nucleotide-binding domain 2) of SUR but appeared to involve complex allosteric interactions between transmembrane and nucleotide-binding domains. In addition to providing direct evidence for the structure-function relationship governing K(ATP) channel activation by potassium channel-opening drugs, a family of drugs of the highest therapeutic interest, these findings delineate the determinants of ligand specificity within the modular ATP-binding cassette-transporter architecture of SUR.     

Glimepiride block of cloned beta-cell, cardiac and smooth muscle K(ATP) channels

1. We examined the effect of the sulphonylurea glimepiride on three types of recombinant ATP-sensitive potassium (K(ATP)) channels. 2. K(ATP) channels share a common pore-forming subunit, Kir6.2, which associates with different sulphonylurea receptor isoforms (SUR1 in beta-cells, SUR2A in heart and SUR2B in smooth muscle). 3. Kir6.2 was coexpressed with SUR1, SUR2A or SUR2B in Xenopus oocytes and macroscopic K(ATP) currents were recorded from giant inside-out membrane patches. Glimepiride was added to the intracellular membrane surface. 4. Glimepiride inhibited Kir6.2/SUR currents by interaction with two sites: a low-affinity site on Kir6.2 (IC(50)= approximately 400 microM) and a high-affinity site on SUR (IC(50)=3.0 nM for SUR1, 5.4 nM for SUR2A and 7.3 nM for SUR2B). The potency of glimepiride at the high-affinity site is close to that observed for glibenclamide (4 nM for SUR1, 27 nM for SUR2A), which has a similar structure. 5. Glimepiride inhibition of Kir6.2/SUR2A and Kir6.2/SUR2B currents, but not Kir6.2/SUR1 currents, reversed rapidly. 6. Our results indicate that glimepiride is a high-affinity sulphonylurea that does not select between the beta-cell, cardiac and smooth muscle types of recombinant K(ATP) channel, when measured in inside-out patches. High-affinity inhibition is mediated by interaction of the drug with the sulphonylurea receptor subunit of the channel.     

Different molecular sites of action for the KATP channel inhibitors,PNU-99963 and PNU-37883A

1. We investigated the mechanism of action of two novel nonsulphonylurea ATP-sensitive potassium channel (K(ATP)) inhibitors, PNU-99963 and PNU-37883A, on four types of cloned K(ATP) channels. 2. Whole-cell currents were recorded in a symmetrical potassium (140 mM) gradient in HEK-293 cells stably expressing Kir6.2/SUR1, Kir6.2/SUR2A, Kir6.2/SUR2B or Kir6.1/SUR2B. 3. PNU-99963 potently inhibited the four K(ATP) channel clones. The concentration at which half-maximum current was inhibited (IC(50)) was 66, 41, 43 and 11 nM for Kir6.2/SUR1, Kir6.2/SUR2A, Kir6.2/SUR2B and Kir6.1/SUR2B, respectively. In contrast, PNU-99963 up to a concentration of 3 microM had no significant effect on current generated in HEK-293 cells by transiently expressing Kir6.2Delta26, a C-terminal truncated pore-forming subunit of Kir6.2. 4. PNU-37883A inhibited four types of K(ATP) channels, but to different extents. Inhibition of the putative smooth muscle K(ATP) channel types, Kir6.2/SUR2B (IC(50); 15 microM) and Kir6.1/SUR2B (IC(50); 6 microM), was significantly greater than inhibition of either the pancreatic beta cell or cardiac K(ATP) channel clones. Moreover, PNU-37883A significantly inhibited currents generated by expressing Kir6.2Delta26 alone, with an IC(50) of 5 microM, which was significantly increased to 38 microM when Kir6.2Delta26 was expressed with SUR2B. 5. In conclusion, two structurally different nonsulphonylurea compounds, PNU-99963 and PNU-37883A, inhibit K(ATP) channels via different mechanisms, namely through the sulphonylurea receptor (SUR) and the pore-forming subunits, respectively, although SUR2B reduced the inhibitory effect of PNU-37883A. While PNU-99963 potently inhibits all the four cloned K(ATP) channels, PNU-37883A has a degree of selectivity towards both smooth muscle K(ATP) channels, but could not discriminate between them.     

Molecular and functional diversity of ATP-sensitive K+ channels: the pathophysiological roles and potential drug targets

ATP-sensitive K(+) (K(ATP)) channels comprise the pore-forming subunit (Kir6.1 or Kir6.2) and the regulatory subunit sulfonylurea receptors (SUR1 or SUR2). K(ATP) channels with different combinations of these subunits are present in various tissues and regulate cellular functions. From the analysis of mouse models with targeted deletion of the gene encoding the pore-forming subunit Kir6.1 or Kir6.2, functional roles of K(ATP) channels in various organs have been clarified. Kir6.1(-/-) mice showed sudden death associated with ST elevation and atrioventricular block in ECG, a phenotype resembling Prinzmetal angina in humans. Kir6.2(-/-) mice were more susceptible to generalized seizure during hypoxia than wild-type (WT) mice, suggesting that neuronal K(ATP) channels, probably composed of Kir6.2 and SUR1, play a crucial role for the protection of the brain against lethal damage due to seizure. In Kir6.2(-/-) mice lacking the sarcolemmal K(ATP) channel activity in cardiac cells, ischemic preconditioning failed to reduce the infarct size, suggesting that sarcolemmal K(ATP) channels play an important role in cardioprotection against ischemia/reperfusion injuries in the heart. Mitochondrial K(ATP) channels have been also proposed to play a crucial role in cardioprotection, although the molecular identity of the channel has not been established. Nicorandil and minoxidil, K(+) channel openers activating mitochondrial K(ATP) channels, decreased the mitochondrial membrane potential, thereby preventing the Ca(2+) overload in the mitochondria of guinea-pig ventricular cells. SURs are the receptors for K(+) channel openers and the activating effects on sarcolemmal K(ATP) channels in cardiovascular tissues could be modulated by the interaction of nucleotides. Due to the molecular diversity of the accessory and pore subunits of K(ATP) channels, there would be considerable differences in the tissue selectivity of K(ATP) channel-acting drugs. Studies of Kir6.1 and Kir6.2 knockout mice indicate that K(ATP) channels are involved in the mechanisms of the protection against metabolic stress. Further clarification of physiological as well as pathophysiological roles of K(ATP) channels may lead to a new therapeutic strategy to improve the quality of life.     

Mg2+ sensitizes KATP channels to inhibition by DIDS: dependence on the sulphonylurea receptor subunit

1. ATP-sensitive potassium channels (K(ATP) channels) consist of pore-forming Kir6.x subunits and of sulphonylurea receptors (SURs). In the absence of Mg(2+), the stilbene disulphonate, DIDS, irreversibly inhibits K(ATP) channels by binding to the Kir subunit. Here, the effects of Mg(2+) on the interaction of DIDS with recombinant K(ATP) channels were studied in electrophysiological and [(3)H]-glibenclamide binding experiments. 2. In inside-out macropatches, Mg(2+) (0.7 mM) increased the sensitivity of K(ATP) channels towards DIDS up to 70 fold (IC(50)=2.7 micro M for Kir6.2/SUR2B). Inhibition of current at DIDS concentrations > or =10 micro M was irreversible. 3. Mg(2+) sensitized the truncated Kir6.2Delta26 channel towards inhibition by DIDS only upon coexpression with a SUR subunit (SUR2B). The effect of Mg(2+) did not require the presence of nucleotides. 4. [(3)H]-glibenclamide binding to SUR2B(Y1206S), a mutant with improved affinity for glibenclamide, was inhibited by DIDS. The potency of inhibition was increased by Mg(2+) and by coexpression with Kir6.2. 5. In the presence of Mg(2+), DIDS inhibited binding of [(3)H]-glibenclamide to Kir6.2/SUR2B(Y1206S) with IC(50)=7.9 micro M by a non-competitive mechanism. Inhibition was fully reversible. 6. It is concluded that the binding site of DIDS on SUR that is sensed by glibenclamide does not mediate channel inhibition. Instead, Mg(2+) binding to SUR may allosterically increase the accessibility and/or reactivity of the DIDS site on Kir6.2. The fact that the Mg(2+) effect does not require the presence of nucleotides underlines the importance of this ion in modulating the properties of the K(ATP) channel.     

Inhibition of ATP-sensitive K+ channels by substituted benzo[c]quinolizinium CFTR activators

The substituted benzo[c]quinolizinium compounds MPB-07 and MPB-91 are novel activators of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel. High homologies between CFTR and the sulfonylurea receptor (SUR), which associates with the potassium channel Kir6.2 to form the ATP-sensitive K(+) (K(ATP)) channel, prompted us to examine possible effects of these compounds on K(ATP) channels using electrophysiological recordings and binding assays. Activity of recombinant K(ATP) channels expressed in Xenopus oocytes was recorded in the inside-out configuration of the patch-clamp technique. Channels were practically unaffected by MPB-07 but were fully blocked by MPB-91 with half-inhibition achieved at approximately 20 microM MPB-91. These effects were similar on channels formed by Kir6.2, and either the SUR1 or SUR2A isoforms were independent of the presence of nucleotides. They were not influenced by SUR mutations known to interfere with its nucleotide-binding capacity. MPB-91, but not MPB-07, was able to displace binding of glibenclamide to HEK cells expressing recombinant SUR1/Kir6.2 channels. Glibenclamide binding to native channels from pancreatic MIN6 cells was also displaced by MPB-91. A Kir6.2 mutant able to form channels without SUR was also blocked by MPB-91, but not by MPB-07. These observations demonstrate that neither MPB-07 nor MPB-91 interact with SUR, in spite of its high homology with CFTR, and that MPB-91 blocks K(ATP) channels by binding to the Kir6.2 subunit. Thus, caution should be exercised when planning to use MPB compounds in cystic fibrosis therapy, specially MPB-91 which could nonetheless find interesting applications as the precursor of a new class of K channel blockers.     

Characterization of K(ATP)-channels in rat basilar and middle cerebral arteries: studies of vasomotor responses and mRNA expression

Changes in the activity of K+ channels represent a major mechanism that regulates vascular tone. Cerebrovascular adenosine 5'-triphosphate-sensitive K+(K(ATP)) channels were characterized in studies of the molecular expression and vasomotor reactivity to different K(ATP) channel openers in rat basilar and middle cerebral arteries. Both arteries showed strong mRNA expression of the subunits of the pore-forming inward-rectifying K+ channel type 6.1 (Kir6.1), Kir6.2 and the connected sulfonylurea receptor (SUR) subunits, SUR1 and SUR2B, while only weak bands for SUR2A were seen. The K(ATP) channel openers induced relaxation of prostaglalndin F2alpha-precontracted isolated basilar and middle cerebral arteries with the order of potency N-Cyano-N-(1,1-dimethylpropyl)-N'-3pyridylguanidine (P-1075)>levcromakalim>N-(4-Phenylsulfonylphenyl)-3,3,3-trifluoro-2-hydroxy-2-methylpropanamide (ZM226600)>pinacidil>diazoxide. The responses induced by levcromakalim, ZM226600 and diazoxide were significantly more potent in basilar arteries than in middle cerebral arteries, while pinacidil and P-1075 were equipotent. Endothelium removal decreased (P<0.05) the sensitivity (pIC50) of basilar arteries, but not of middle cerebral arteries, to pinacidil, levcromakalim, P-1075 and ZM226600. The maximum relaxant response to P-1075 was stronger (P<0.005) in basilar arteries with endothelium than without endothelium. Correlation of the relaxant potency of K(ATP) channel openers in rat basilar and middle cerebral arteries with historical measurements of affinity obtained in COS-7 cell lines expressing either SUR1, SUR2A or SUR2B showed that vasodilatation by K(ATP) channel openers correlated with binding to either the SUR2A or the SUR2B subunit. Glibenclamide was a blocker of relaxation induced by pinacidil, levcromakalim, P-1075 and ZM226600 in basilar arteries. Only a weak antagonistic effect of glibenclamide on pinacidil-, levcromakalim- and ZM226600-induced relaxations was found in middle cerebral arteries. The subunit profile and the observed pharmacological properties suggest that the K(ATP) channels expressed in rat basilar and middle cerebral artery are likely to be composed of SUR2B co-associated with Kir6.1 or Kir6.2. In basilar arteries, but not in middle cerebral arteries, endothelial K(ATP) channels may be involved.